Renal ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) in many clinical settings including cardiovascular surgery, sepsis, and kidney transplantation. Ischemic AKI is associated with increased morbidity, mortality, and prolonged hospitalization (1, 2).
Acute ischemia leads to depletion of adenosine triphosphate (ATP), inducing tubular epithelial cell (TEC) injury, and hypoxic cell death. Reperfusion further amplifies injury by promoting the formation of reactive oxygen species (ROS), and inducing leukocyte activation, infiltration, and inflammation (3-6).
Multiple studies have shown the important role of iron and ROS in mediating apoptotic and necrotic cell death and the ensuing inflammatory response during the course of IRI (7, 8). The ischemic stage of the injury results in mitochondrial membrane depolarization, DNA fragmentation, translocation of cytochromes from the mitochondria into the cytosol (9, 10) and breakdown of heme proteins like the cytochrome c results in a reduction of heme associated ferric (Fe3+) iron to ferrous (Fe2+) iron and thereby increase the levels of catalytically active or labile form of iron (11-13). A key pathological characteristic of labile iron (Fe2+) is its ability to catalyze the generation of tissue damaging hydroxyl radical (OH—) by an interaction with superoxide anion (O−2) and hydrogen peroxide (H2O2) via Haber-Weiss reaction, both of which are increased during IRI (7, 14). Labile iron's contribution to oxidative stress and cellular damage has been demonstrated not only in renal ischemia reperfusion injury (15, 16) but also in other models of AKI (17, 18) and iron chelation with desferrioxamine induces protection in diverse animals models of AKI (19).
Iron (Fe2+) is exported from the cells by the only known iron export protein, ferroportin (20). Ferroportin is significantly expressed on macrophages, hepatocytes, renal proximal and distal tubular cells, and enterocytes (21-23). Intracellular iron levels regulate ferroportin expression; high intracellular iron induces ferroportin-mediated iron export into circulation (20). To sustain physiologic iron requirement yet avoid iron toxicity, an endogenous peptide hormone Hepcidin (HAMP), primarily produced by hepatocytes (23, 24), and regulates systemic iron balance. The main known function of HAMP is to covalently modify ferroportin, which leads to its internalization and lysosomal degradation, and thereby prevent cellular iron egress (25, 26). HAMP is acutely and positively regulated during iron imbalance (25), inflammation (27, 28) and has antibacterial properties (29, 30; see also 60 and 61). Hypoxia, however, negatively regulates it (31). While human studies have indicated a positive correlation between increased urinary Hepcidin levels and protection against AKI, a direct pathogenic role of hepcidin has not been examined in any model of AKI (32).
Mature hepcidin is a 25 amino acid (a.a.) residue peptide. Its production appears to be regulated at the transcriptional level and major stimuli regulating hepcidin production include iron and the regulatory signals pertaining to erythropoietic demands for iron. Hepcidin is also an acute-phase reactant and increases during inflammation. Other hepcidin regulators include hepatocyte growth factor (HGF), epidermal growth factor (EGF), steroid hormones (estrogen, testosterone), and metabolic pathways (starvation/gluconeogenesis). Hepcidin production increases in response to iron loading and this prevents further absorption of dietary iron and the development of iron overload. Plasma iron and liver iron stores regulate hepcidin transcription. Both serum iron and liver iron accumulation activate the BMP receptor and its Smad1/5/8 pathway, and increase hepcidin mRNA concentrations in hepatocytes. The BMP co-receptor hemojuvelin (HJV) is also required for this response.
For extracellular iron, transferrin receptors 1 and 2 (TfR1 and TfR2) are the likely sensors of holo-transferrin concentrations. At higher holo-Tf concentrations, HFE is displaced from TfR1 and associates with TfR2. HFE and TfR2 in turn may interact with HJV, thus potentiating BMP signaling.
Mutations in Hfe, TfR2, Hjv, Bmp6, BMP receptors Alk2 and Alk3, and Smad4 all impair hepcidin regulation by iron. Hepcidin production is further modulated by the transmembrane serine protease TMPRSS6, also known as matriptase-2, and by neogenin, a multifunctional transmembrane receptor. It has been proposed that these proteins act by post-translationally regulating the levels of membrane-associated HJV. The specific involvement of these proteins in iron sensing is also uncertain.
Hepcidin is suppressed in conditions associated with increased erythropoietic activity. Hemorrhage, hemolysis, and injections of erythropoietin all result in a rapid decrease in hepcidin. In anemias with ineffective erythropoiesis, hepcidin levels are chronically suppressed. This is thought to be the cause of iron overload in nontransfused patients. Hepcidin increases rapidly following inflammatory and infectious stimuli via the IL-6 pathway. Because hepcidin deficiency or excess plays important roles in the pathogenesis of various iron disorders, hepcidin agonists and antagonists may be potentially useful in clinical practice.
Hepcidin agonists such as PR73 (a minihepcidin) are compounds that can mimic the function of hepcidin or potentiate its endogenous synthesis and may be able to prevent systemic accumulation of iron (See Ganz et al., International Pat. Pub. WO2013086143 A1). Such compounds may provide additional treatment options for patients who do not respond well to standard treatment regimens. Minihepcidins are peptide-based hepcidin agonists that were rationally designed based on the region of hepcidin that interacts with ferroportin. A nine amino acid N-terminal fragment of hepcidin (DTHFPICIF) is crucial for its hormonal activity. This particular fragment was further engineered: unnatural amino acids (N-substituted and β-homo amino acids) were introduced to increase resistance to proteolysis, and fatty acids were conjugated to prolong the half-life in circulation. This yielded analogs that are at least as potent as full-length hepcidin and have a longer duration of action. One such analog, the minihepcidin PR65, was tested in hepcidin knockout mice, a model of severe hemochromatosis. Treatment prevented the development of iron overload in non-overloaded hepcidin knockout mice. Treatment of mice with pre-existing iron overload was less effective but still led to partial redistribution of iron from the liver to the spleen within 2 weeks. At high doses, PR65 caused profound iron restriction and anemia, indicating that minihepcidin therapy will likely require titration to effect to avoid excessive hypoferremia and iron restriction.
Hepcidin production can be increased by antagonizing TMPRSS6, a negative regulator of hepcidin. Homozygous inactivation of Tmprss6 in thalassemic th3/+ mice increased hepcidin levels, ameliorated iron overload, and improved ineffective erythropoiesis. Targeting Tmprss6 with RNA-based therapeutics such as antisense oligonucleotides (ASOs) and siRNAs against Tmprss6 was effective in a mouse model of iron overload.
Hepcidin production can also be stimulated by BMP6 and its agonists. In patients undergoing low-molecular-weight heparin therapy to prevent deep vein thrombosis, serum hepcidin concentrations decreased by ˜80% within 2-5 days after the start of the treatment. This was associated with increased serum iron and transferrin saturation. Heparin itself is an anti-inflammatory agent, which may be a contributory factor in its anti-hepcidin activity. HJV, a BMP co-receptor essential for hepcidin expression, is another molecular target that can be exploited to interfere with hepcidin production. Membrane-linked HJV and its soluble form (shave) have opposing effects on hepcidin expression, and shave decreases Smad signaling and hepcidin levels. Soluble HJV-Fc fusion protein (sHJV.Fc) ameliorated anemia of inflammation (AI) in a rat model in which AI was induced with group A streptococcal peptidoglycan-polysaccharide (PG-APS). Four-week therapy resulted in increased hemoglobin and serum iron, although hepcidin mRNA had not significantly decreased by this point.
LDN-193189, a derivative of dorsomorphin which specifically antagonizes the kinase activity of BMP receptor isotypes ALK2, ALK3, and ALK6, effectively reversed anemia in the rat model of AI caused by PG-APS.
Inflammation induces hepcidin expression via IL-6-Stat3 and possibly other pathways and neutralizing monoclonal antibodies directed against IL-6 or IL-6 receptors can be used to decrease hepcidin synthesis in animal models and humans with inflammatory conditions.
There is a long felt need in the art for compositions and methods useful for preventing and treating acute kidney injury associated with renal ischemia reperfusion, including during surgery such as transplant surgery. The present invention satisfies these needs.